Fusion proteins for inhibiting angiogenesis

ABSTRACT

The present invention relates to a biologic that inhibits angiogenesis. In particular, the present invention relates to fusion proteins that inhibit the integrin activated pathway and one other angiogenic factor-activated pathway, the compositions of these fusion proteins, as well as methods for producing and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/739,089 filed Dec. 21, 2017, which is a 371 ofInternational Application No. PCT/IB2016/053794 filed Jun. 24, 2016,which claims the benefit of U.S. Provisional Application No. 62/185,716filed Jun. 28, 2015, the disclosures of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a biologic that inhibits angiogenesis.In particular, the present invention relates to fusion proteins thatinhibit angiogenic factor-activated pathways, the compositions of thesefusion proteins, as well as methods for producing and using the same.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named688947_20U2_Sequence_Listing.txt, created on Feb. 18, 2020, and having asize of 37 kb, and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Angiogenesis is the process of growing new blood vessels from theexisting vasculature. It plays an important role in severalphysiological processes, including embryonic development, as well astissue and wound repair (Folkman J et al., Angiogenic factors. Science1987; 235:442-7). The physiologic steps of angiogenesis are wellcharacterized, and involve proteolysis of the extracellular matrix,proliferation, migration, and assembly of the endothelial cells into atubular channel, mural cell recruitment and differentiation, andextracellular matrix production (Carmeliet P et al., Nature. 2011;473:298-307). Pathologic angiogenesis may occur in tumor formation,ocular disorders (e.g., diabetic retinopathy, diabetic macular edema ormacular degeneration) arthritis, psoriasis, fibrotic diseases,inflammatory diseases, and arteriosclerosis (Polverini P J. Crit RevOral Biol Med. 1995; 6(3):230-47).

Pathologic angiogenesis is more heterogeneous and chaotic, oftendemonstrating tortuous vessel organization, hypoxic voids of varioussizes, uneven and imperfect vessel walls and linings, and ineffectiveperfusion (Jain R K., Nat Med. 2003; 9(6):685-93). These distinctcharacteristics of new blood vessel formation in diseases have madetherapeutic targeting of angiogenesis a challenge. Although anti-VEGFtherapies such as Lucentis®, Eylea®, or off-label use of Avastin® cangenerally stabilize or improve visual function, sub-retinal scarring(fibrosis) can develop in approximately half of all treated eyes withintwo years after anti-VEGF treatment and has been identified as one causeof unsuccessful outcomes (Daniel E et al., Ophthalmology. 2014;121(3):656-66). Many of the critical players in sub-retinal fibrosis arelikely to be the growth factors and the matricellular proteins that areinvolved in the fibrotic process (cell proliferation, migration and ECMremodeling). Despite its complexity, with our increasing knowledge ofthe angiogenic process, anti-angiogenic drug development remains an areaof great interest.

Currently, many key players in the neovascularization process have beenidentified, and the vascular endothelial growth factor (VEGF) family hasa predominant role. The human VEGF family consists of 6 members: VEGF-AVEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). Inaddition, multiple isoforms of VEGF-A, VEGF-B, and PlGF are generatedthrough alternative RNA splicing (Sullivan et al., MAbs, 2002, 2(2):165-75). VEGF-A is the primary factor involved with angiogenesis; itbinds to both VEGFR-1 and VEGFR-2. The strategy of inhibitingangiogenesis by obstructing VEGF-A signaling has established successfultherapies for treatment of specific cancers as well as retinalneovacular and ischemic diseases. (Major et al., J Pharmacol Exp Ther.,1997, 283(1):402-10; Willet et al., Nat. Med. 2004, 10:145-7;Papadopoulos et al., Angiogenesis, 2012, 15(2):171-85; Aiello et al.,PNAS, 1995, 92:10457-61).

Other growth factors, cytokines, chemokines include Platelet DerivedGrowth Factors (PDGFs), Transforming Growth Factors beta (TGF-β),Epidermal Growth Factors (EGFs), Nerve Growth Factors (NGFs),Hypoxia-Induced Factor (HIF), Connective-Tissue Growth Factor (CTGF),Granulocyte—Macrophage Colony-Stimulating Factor (GM-CSF), Insulin-LikeGrowth Factor (IGF), Hepatocyte Growth Factors/Scatter Factor (HGF/SF),Tumor Necrosis Factor alpha (TNF-α), Interleukin 1 (IL-1), Interleukin 6(IL-6), Interleukin 8 (IL-8), Interleukin 17 (IL-17), Interleukin 18(IL-18), Interleukin 20 (IL-20), Interleukin 23 (IL-23),Chemoattractants such as C—C motif Ligand (CCL28, CCL21) and C—X—C motifLigand (CXCL1, CXCL5), Macrophage migration Inhibitory Factor (MIF), andimmune cell surface proteins such as Clusters of Differentiation (CDs).These factors are reported to be overespressed and play key roles inangiogenesis-related diseases (Elshabrawy et al., Angiogenesis (2015)18:433-448; Brian P. Eliceiri, Circ Res. 2001 Dec. 7; 89(12):1104-10).Targeting these factors to reduce their downstream pathway activationmay decrease angiogenesis-related diseases.

Integrins, a family of cell surface receptors, are also found to beoverexpressed on the endothelial cell surface and are believed tofacilitate the growth and survival of newly forming vessels duringangiogenesis. Integrins are heterodimeric cell surface receptors thatinteract with extracellular matrix proteins and are critical for manybiological processes. The expression of integrins in various cell typesare involved in tumor progression, and their ability to crosstalk withgrowth factor receptors has made them attractive therapeutic targets.(Staunton D E, et al., Adv Immunol. 2006; 91:111-57; Avraamides, C. J.,et al., Nat Rev Cancer 2008; 8:604-617.) In particular, the integrinαvβ3 is upregulated in both tumor cells and angiogenic endothelialcells, and is important for tumor cell migration, angiogenesis, anddysregulated cell signaling. Therefore, antagonists of the integrin αvβ3are intensively studied for their anti-angiogenic and anti-tumorproperties (Desgrosellier J S et al., Nat Rev Cancer. 2010 10:9-22).

Disintegrins are the peptides found in snake venom of the viper familyand mainly inhibit the function of β1- and β3-associated integrins. Theywere first identified as inhibitors of integrin αIIbβ3 and weresubsequently shown to bind with high affinity to other integrins,blocking the interaction of integrins with RGD-containing proteins. Theycontain 47 to 84 amino acids with about 4 to 7 disulfide bonds and carrythe same RGD motif (McLane M A, et al., Proc Soc Exp Biol Med 1998 219:109-119; Niewiarowski S, et al., Semin Hematol 1994 31: 289-300; CalveteJ J., Curr Pharm Des. 2005 11: 829-835; Blobel C P et al., Curr OpinCell Biol 1992 4: 760-765). The conserved RGD sequence in thedisintegrin family plays the most important role in recognizing theintegrins. Disintegrins were found to interact with eight out oftwenty-four integrins and inhibited integrin-mediated cell adhesion,migration, and angiogenesis (McLane M A, et al., Front Biosci. 2008 13:6617-6637; Swenson S, et al., Curr Pharm Des. 2007 13: 2860-2871).Animal studies showed that disintegrins targeted neovascular endotheliumand metastatic tumors, indicating their potential use in cancer therapy.The specific binding of RGD-containing proteins to integrin is afunction of both the conformation and the local sequence surrounding theRGD motif. Many studies have shown that the residues flanking the RGDmotif of RGD-containing proteins affect their binding specificities andaffinities to integrins (Scarborough R M et al., J Biol Chem 1993 268:1058-1065; Rahman S et al., Biochem J 1998 335: 247-257).

Angiogenesis is a complex biological process which involves variousgrowth factors and signaling receptors, and targeting single moleculesin the signaling cascade may not provide an effective clinical treatmentfor uncontrolled angiogenesis in diseases such as cancer. Therefore,there is a growing need to develop innovative therapeutics capable ofbinding several key angiogenic factors in a cooperative manner toeffectively inhibit angiogenesis and progression of the disease.

BRIEF SUMMARY OF THE INVENTION

The invention provided herein discloses polypeptides for specificallybinding to multiple targets so as to antagonize several angiogenicfactors. The invention also provides fusion proteins that inhibit aselective integrin pathway and other angiogenic pathways. The inventionfurther provides compositions having these fusion proteins. Theinvention further provides compositions having a vector that comprises anucleic acid encoding the fusion proteins. The invention furtherdescribes methods for producing and using these fusion proteins for thetreatment or prevention of angiogenic diseases, ocular diseases,autoimmune diseases, inflammatory diseases, fibrotic diseases, and/orcancer.

In accordance with the present invention, a fusion protein comprising anintegrin binding peptide selected from a group consisting ofdisintegrin, anti-integrin αvβx antibody, anti-integrin α5β1 antibody,fibronectin targeting integrin αvβx or α5β1 and their integrin bindingfragments, other protein binding peptide targeting an angiogenic factorand a Fc domain, wherein x is 1, 3, 5, 6 or 8. In some embodiments, theangiogenic factor comprises Angiopoietin (ANG), Ephrin (Eph), FibroblastGrowth Factor (FGF), Neuropilin (NRP), Plasminogen Activators,Platelet-Derived Growth Factor (PDGF), Tumor Growth Factor beta (TGF-β),Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial cadherin(VE-cadherin), Epidermal Growth Factors (EGFs), Nerve Growth Factors(NGFs), Connective-Tissue Growth Factor (CTGF), Granulocyte MacrophageColony-Stimulating Factor (GM-CSF), Insulin-like Growth Factor (IGF),Hepatocyte Growth Factors/Scatter Factor (HGF/SF), Tumor Necrosis Factoralpha (TNF-α), Interleukin 1 (IL-1), Interleukin 6 (IL-6), Interleukin 8(IL-8), Interleukin 17 (IL-17), Interleukin 18 (IL-18), Interleukin 20(IL-20), Interleukin 23 (IL-23), Chemoattractants such as C—C motifLigand (CCL28, CCL21), C—X—C motif Ligand (CXCL1, CXCL5), Macrophagemigration Inhibitory Factor (MIF), immune cell surface protein such asClusters of Differentiation (CDs), and receptors thereof.

According to one aspect, the invention provides a fusion proteincomprising an integrin binding peptide having an amino acid sequenceselected from a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, oramino acid sequence having at least 85% sequence identity to SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,and SEQ ID NO: 7, other protein binding peptide comprising extracellulardomains of VEGF receptors, and a Fc domain, wherein the integrin bindingpeptide has at least one mutation on or adjacent to a RGD motif.

According to another aspect, the invention provides a fusion proteincomprising an integrin binding peptide that includes disintegrin and itsintegrin binding fragments, other protein binding peptide comprisingextracellular domains of VEGF receptors and a Fc domain, wherein theintegrin binding peptide comprises at least one mutation on or adjacentto the RGD motif. In some embodiments, the fusion protein comprises theintegrin binding peptide, the Fc domain, and the other protein bindingpeptide from C-terminus to N-terminus. In some embodiments, the fusionprotein comprises the integrin binding peptide, the Fc domain, and theother protein binding peptide from N-terminus to C-terminus. In furtherembodiments of the fusion protein, the integrin binding peptide has atleast 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

In accordance with other embodiments, the invention also provides afusion protein comprising an integrin binding peptide that includes anamino acid sequence of SEQ ID NO:1 with at least one mutation on oradjacent to the RGD motif, an amino acid sequence having at least 85%sequence identity to SEQ ID NO: 1 or an amino acid sequence of SEQ IDNO: 2, a human or humanized constant sub-region comprising animmunoglobulin CH2 domain and a CH3 domain, and other protein bindingpeptide having an Ig-like domain D2 of a VEGFR1 and an Ig-like domain D3of a VEGFR2. In a further embodiment of the fusion protein, the integrinbinding peptide has at least 85% sequence identity to SEQ ID NO: 2.

In accordance with one aspect, the invention provides a nucleic acidsequence encoding a fusion protein of the invention disclosed herein.

In accordance with another aspect, the invention provides a dimer offusion proteins of the invention disclosed herein.

In accordance with another aspect, the invention provides a method ofproducing a fusion protein of the invention, comprising culturing a hostcell transfected with a vector comprising a nucleic acid sequence of theinvention, under a condition that produces the fusion protein andrecovering the fusion protein produced by the host cell.

In accordance with another aspect, the invention provides a method oftreating or preventing an angiogenic disease comprising administering aneffective amount of the fusion protein to a subject in need thereof. Insome embodiments, the angiogenic disease comprises rheumatoid arthritis,inflammatory arthritis, osteoarthritis, ocular and systemic cancer,tumor related metastasis and invasion, systemic fibrotic diseasesincluding idiopathic lung fibrosis (IPF), nonalcoholic steatohepatitis(NASH) or liver fibrosis, diabetic nephropathy and/or kidney fibrosis,dermal fibrosis or keloid, wound healing, cardio-fibrosis, andischemia-induced stroke; ocular disease characterized byneovascularization or ischemia (such as choroidal neovascularization),uveitis, retinitis pigmentosa, age-related macular degeneration (AMD),diabetic retinopathy and diabetic macular edema (DME), central andbranch retinal vein occlusion (CRVO and BRVO), human retinopathy ofprematurity (ROP), polypoidal choroidal vasculopathy (PCV), symptomaticvitreomacular adhesion, and glaucoma. In some embodiments, the fusionprotein of the invention comprises a linker sequence between theintegrin binding peptide and the other protein binding peptide. In someembodiments, the fusion protein of the invention comprises the signalpeptide sequence upstream of the other binding domain.

In accordance with another aspect, the invention provides a compositioncomprising a fusion protein of the invention and a pharmaceuticallyacceptable carrier.

In accordance with another aspect, the invention provides a polypeptidetargeting to multiple angiogenic factors comprising: an integrin bindingpeptide comprising an amino acid sequence selected from a groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or amino acid sequencehaving at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7;other protein binding peptide selected from a group consisting ofextracellular domains of VEGF receptors, anti-VEGF antibody, anti-PDGFantibody and their fragments; and a Fc domain; wherein the integrinbinding peptide comprising at least one mutation on or adjacent to theRGD motif.

In some embodiments, the extracellular domains of VEGF receptors in thepolypeptide of the invention comprise the Ig-like domains D1-D7 of theVEGF receptors. In some embodiments, the extracellular domains of VEGFreceptors in the polypeptide of the invention comprise: i) the Ig-likedomain 2 (D2) of a VEGFR1 and an Ig-like domain 3 (D3) of a VEGFR2; ii)the amino acid sequence of SED ID NO: 10; or iii) an amino acid sequencehaving at least 90% identity to SEQ ID NO: 10. In some embodiments, theextracellular domains of PDGF receptors in the polypeptide of theinvention comprise the Ig-like domains 1-5 of the PDGF receptors. Insome embodiments, the extracellular domains of PDGF receptors in thepolypeptide of the invention comprise: i) the Ig-like domains 1-3 of aPDGFRβ; ii) the amino acid sequence of SED ID NO: 11; or iii) an aminoacid sequence having at least 90% identity to SEQ ID NO: 11. In someembodiments, the polypeptide of the invention further comprises aGly-Ser (GS) or Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (G₉) linker betweenthe Fc domain and the integrin binding peptide or the extracellulardomains of VEGF or PDGF receptors.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofexemplary embodiments will become more apparent and may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings.

FIG. 1 is a schematic illustrating the composition of an exemplaryfusion protein according to one embodiment of the invention.

FIG. 2 is a schematic illustrating the purified Fusion Protein 1analyzed by SDS-PAGE.

FIG. 3 is a schematic illustrating a VEGF-ligand binding assay.

FIG. 4 is a schematic illustrating a αvβ3 competitive binding assay.

FIG. 5 is a schematic illustrating VEGF-induced HUVEC proliferation.

FIG. 6 is a schematic illustrating the inhibition of CHO-αvβ3 celladhesion.

FIGS. 7A-G is a schematic illustrating tube formation inhibitory effectby Fusion Proteins.

FIGS. 8A-D provide one embodiment showing the dose-response inhibitionof VEGF-induced leakage in Dutch Belted Rabbits by Fusion Proteins.

FIGS. 9A-C provide one embodiment showing the reduction of lesion sizein laser-induced choroidal neovascularization (CNV) in rats by FusionProteins. An arrow (Y) denotes location of laser lesion.

DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person skilled in theart to which this invention belongs.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a binding domain” includes a plurality of bindingdomains and equivalents thereof known to those skilled in the art.

As used herein, the term “polypeptide” and “protein” may be usedinterchangeably to refer to a long chain of peptide having an amino acidsequence of the native protein or the amino acid sequence with one ormore mutations such as deletion, additions, and/or substitutions of oneor more amino acid residues.

A “fusion protein” refers to a protein having two or more portionscovalently linked together, where each of the portions is derived fromdifferent proteins.

The present invention provides a fusion protein comprising an integrinbinding peptide selected from a group consisting of disintegrin (seeU.S. Pat. No. 7,943,728 and PCT Application No. PCT/US 15/46322 for thedescription of amino acid sequences, each of which is incorporated byreference in its entirety), anti-integrin αvβx antibody (see U.S. Pat.Nos. 6,160,099 and 8,350,010 for the description of amino acidsequences, each of which is incorporated by reference in its entirety),anti-integrin α5β1 antibody, fibronectin (see U.S. Pub. No. 2015/0218251for the description of amino acid sequences, which is incorporated byreference in its entirety) targeting integrin isoform αvβx or α5β1 andtheir integrin binding fragments, other protein binding peptidetargeting an angiogenic factor and a Fc domain, wherein x is 1, 3, 5, 6or 8.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavychains and two light chains inter-connected by disulfide bonds. Afull-length heavy chain includes a variable region domain, VH, and threeconstant region domains, CH1, CH2 and CH3. The VH domain is at theamino-terminus of the polypeptide, and the CH3 domain is at thecarboxy-terminus. A full-length light chain includes a variable regiondomain, VL, and a constant region domain, CL. An antigen bindingfragment (Fab) is comprised of one light chain and the CH1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule. A Fab′ fragmentcontains one light chain and one heavy chain that contains more of theconstant region, between the CH1 and CH2 domains, such that aninterchain disulfide bond can be formed between two heavy chains to formdiabodies. A variable fragment (Fv) region comprises the variableregions from both the heavy and light chains, but lacks the constantregions. Single-chain fragments (scFv) are Fv molecules in which theheavy and light chain variable regions have been connected by a flexiblelinker to form a single polypeptide chain which forms an antigen-bindingregion. Single chain antibodies are discussed in detail in WO88/01649and U.S. Pat. Nos. 4,946,778 and 5,260,203. As used herein, the term“antibody” includes the an immunoglobulin molecule with two full lengthL-chains and two full length H-chains, and fragments thereof, such as anantigen binding fragment (Fab), a Fv region, a scFv, etc.

The term “Fc domain” refers to a molecule or sequence comprising thesequence of a non-antigen binding portion of an antibody, whether inmonomeric or multimeric form. The original immunoglobulin source of anFc is preferably of human origin and can be from any isotype, e.g., IgG,IgA, IgM, IgE or IgD. A full-length Fc consists of the following Igheavy chain regions: CH1, CH2 and CH3, wherein the CH1 and CH2 regionsare typically connected by a flexible hinge region.

The present invention provides a fusion protein comprising an integrinbinding peptide that includes disintegrin and its integrin bindingfragments, other protein binding peptide comprising extracellulardomains of VEGF receptor and a Fc domain, wherein the integrin bindingpeptide comprises at least one mutation on or adjacent to the RGD motif.In accordance with embodiments of the present invention, the disintegrinand its integrin binding fragments have an amino acid sequence selectedfrom a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or amino acidsequence having at least 85% sequence identity to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQID NO: 7

As used herein, “disintegrin” refers to a class of cysteine-richproteins or polypeptides that are potent soluble ligands of integrins.The RGD motif is a tri-peptide (Arg-Gly-Asp) conserved in most ofmonomeric disintegrins and is located at an integrin-binding loop. Thedisintegrins described herein are isolated from snake venom or derivedfrom wild-type forms and have at least one mutation on or adjacent to aRGD motif to selectively bind to or target to various integrin isoforms.The term “adjacent to a RGD motif” as used herein means any mutationwhich occurs at any amino acid residue within 15-20 amino acids from theRGD motif in a given peptide or polypeptide sequence.

Other amino acid sequence variants of the disintegrin are alsocontemplated. For example, binding affinity and/or other biologicalproperties of a disintegrin can be improved by altering the amino acidsequence encoding the protein. Disintegrin mutants can be prepared byintroducing appropriate modifications into the nucleic acid sequenceencoding the protein or by introducing modification by peptidesynthesis. Such modifications include mutations such as deletions from,insertions into, and/or substitutions within the nucleic or amino acidsequence of the disintegrin. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final amino acid construct ofthe disintegrin provided that the final construct possesses the desiredcharacteristics such as binding to an integrin superfamily member and/orinhibiting the integrin activated pathway.

Substantial modifications in the biological properties of the proteinsor polypeptides are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

A useful method for identifying certain residues or regions of thefusion protein that are preferred locations for mutagenesis is known as“alanine scanning mutagenesis” as described in Science, 1989,244:1081-1085. For example, a residue or group of target residues areidentified (e.g., charged residues such as Arg, Asp, His, Lys and Glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with the target binding partner. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed fusion polypeptide variants arescreened for desired activity. For example, cysteine bond(s) may beadded to the fusion protein or protein components to improve itsstability.

Accordingly, provided herein are disintegrin mutants that can be acomponent of any fusion protein disclosed herein. In some embodiments,the disintegrin comprises an amino acid sequence with at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity to the aminoacid sequence of disintegrins selected from a group consisting ofRhodostomin (SEQ ID NO: 1), Triflavin (SEQ ID NO: 3), Echistatin (SEQ IDNO: 4), Trimucrin (SEQ ID NO: 5), Elegantin (SEQ ID NO: 6) and Trigramin(SEQ ID NO: 7). In some embodiments, a disintegrin comprises an aminoacid sequence having at least one mutation on or adjacent to the RGDmotif of Rhodostomin (SEQ ID NO: 1), Triflavin (SEQ ID NO: 3),Echistatin (SEQ ID NO: 4), Trimucrin (SEQ ID NO: 5), Elegantin (SEQ IDNO: 6) or Trigramin (SEQ ID NO: 7). In some embodiments, the disintegrincomprises an amino acid sequence with at least 85%, at least 90%, atleast 95%, or at least 99% sequence identity to the amino acid sequenceof a disintegrin mutant (SEQ ID NO: 2). The Xaa in SEQ ID NO: 2indicates various positions that can be modified by either insertion,substitution or deletion to produce amino acid sequence variants thatare different from the wild type form of disintegrin. According to someexamples, the Xaa at position 50 of SEQ ID NO: 2 which corresponds toglycine (Gly) in the RGD motif of wild type Rhodostomin (SEQ ID NO: 1)may be substituted with naturally occurring amino acids other thanglycine to generate Rhodostomin mutants. In other examples, one or moreXaa in SEQ ID NO: 2 may also be substituted with naturally occurringamino acids other than those originally found in corresponding positionsof wild type Rhodostomin (SEQ ID NO: 1) to generate various Rhodostominmutants. It is further noted that the disintegrin mutants are notlimited to include only single mutation at any Xaa in SEQ ID NO: 2,multiple mutations that occur at several locations of Xaa in SEQ ID NO:2 or corresponding locations in other consensus sequences of disintegrin(such as SEQ ID NOs: 3-7) may also be encompassed by the scope of theinvention.

Rhodostomin mutants have been described in U.S. Pat. No. 7,943,728 andPCT Application No. PCT/US 15/46322 and their sequences are incorporatedherein by reference. For example, SEQ ID NO: 1 can comprise one or moreof the mutant sequences shown in Table 1 at the indicated amino acidsequence positions.

TABLE 1 SEQ ID Position on SEQ NO Sequence ID NO: 1 24 RIARGDNP 46-53 25RRARGDNP 26 ARGRGDNP 27 ARGRGDDL 28 ARARGDNP 29 KKKRTIC 39-45 30 MKKGTIC31 IEEGTIC 32 KGAGKIC 33 LKEGTIC 34 AKKRTIC 35 KAKRTIC 36 KKARTIC 37KKKATIC 38 KKKRAIC 39 KAKRAIC 40 SKAGTIC 41 KKKRTIC 42 PRWNDL 65-68 43PRNGLYG 44 PGLYG 45 PDLYG 46 PPLYG 47 PRLYG 48 PELYG 49 PYLYG

Although the variants of disintegrins are discussed mostly withreference to the amino acid sequences discussed above, polypeptidesequences or nucleotide sequence encoding the snake venom such asAlbolabrin, Applagin, Basilicin, Batroxostatin, Bitistatin, Cereberin,Cerastin, Crotatroxin, Durissin, Flavoridin, Flavostatin, Halysin,Halystatin, Jararacin, Jarastatin, Kistrin, Lachesin, Lutosin, Molossin,Salmosin, Saxatilin, Tergeminin, Trimestatin, Trimutase, Ussuristatin,Viridian and their mutants having at least one mutation on or adjacentto the RGD motif may also be encompassed by the scope of the presentinvention.

Without being bound by theory, it is contemplated herein thatdisintegrins inhibit the integrin activated pathway by binding to anintegrin superfamily member to block its interaction with a multivalentintegrin receptor. In some aspects, the disintegrin binds to an integrinsuperfamily member which includes but is not limited to the integrinisoforms αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1 and/or αvIIbβ3.

According to the present invention, the other protein binding peptide ofthe fusion protein may be receptor protein that binds to a targetselected from the group consisting of a tumor antigen, a TNF receptorsuperfamily member, a Hedgehog family member, a receptor tyrosinekinase, a proteoglycan-related molecule, a TGF-beta superfamily member,a Wnt-related molecule and an angiogenesis target.

According to some embodiments of the invention, the other proteinbinding peptide may specifically bind to an angiogenesis target whichincludes but is not limited to Angiopoietin (ANG), Ephrin (Eph),Fibroblast Growth Factor (FGF), Neuropilin (NRP), PlasminogenActivators, Platelet-Derived Growth Factor (PDGF), Tumor Growth factorbeta (TGF-β), Vascular Endothelial Growth Factor (VEGF), VascularEndothelial cadherin (VE-cadherin) and their receptors. Therefore, inaccordance with embodiments of the invention, the other protein bindingpeptide may include extracellular portions of a receptor protein thatbinds to and antagonizes the angiogenesis target. In other embodiments,the other protein binding peptide may bind to extracellular portions ofangiogenic factor receptors.

In some embodiments, the other protein binding peptide may be ananti-VEGF antibody (see WO2015/200905 for the description of its aminoacid sequence, which is incorporated herein by reference in itsentirety) that binds to the VEGF ligand or an anti-VEGFR1 or anti-VEGFR2antibody (see U.S. Pat. No. 5,874,542 for the description of its aminoacid sequence, which is incorporated herein by reference in itsentirety) that binds to VEGF receptor. In other embodiments, the otherprotein binding peptide may also be an anti-PDGF antibody (see U.S. Pat.No. 5,094,941 for the description of its amino acid sequence, which isincorporated herein by reference in its entirety) that binds to the PDGFligand or an anti-PDGFR beta antibody (see U.S. Pat. No. 9,265,827 forthe description of its amino acid sequence, which is incorporated hereinby reference in its entirety for all purposes) that binds to PDGFreceptor.

In certain embodiments, the other protein binding peptide binds to thesame VEGF as any one of VEGF receptors (VEGFR): VEGFR1, VEGFR2 andVEGFR3. In some embodiments, the other protein binding peptide comprisesat least one extracellular portion of a VEGFR of any of the VEGFRsdescribed herein. For example, the other protein binding peptidecomprises at least one extracellular portion of VEGFR1 or oneextracellular portion of VEGFR2. In another example, the other proteinbinding peptide comprises one extracellular portion of VEGFR1 such asIg-like domain 2 (D2) and one extracellular portion of VEGFR2 such asIg-like domain 3 (D3). In some aspect, the other protein binding peptidecomprises one extracellular portion of a VEGFR1 comprising amino acidsequence of SEQ ID NO: 8 and one extracellular portion of a VEGFR2comprising amino acid sequence of SEQ ID NO: 9. In some aspect, theother protein binding peptide comprises a fusion of extracellularportions of VEGFR1 and VEGFR2 comprising an amino acid sequence of SEQID NO: 10 or an amino acid sequence with at least 85% sequence identityto SEQ ID NO: 10.

In other embodiments, the other protein binding peptide binds to thesame PDGF as any one of PDGF receptors (PDGFR): PDGFR-α and PDGFR-β. Insome embodiments, the other protein binding peptide comprises at leastone extracellular portion of a PDGFR of any of the PDGFRs describedherein. For example, the other protein binding peptide comprises atleast one extracellular portion of PDGFR-α or one extracellular portionof PDGFR-β. In another example, the other protein binding peptidecomprises one extracellular portion of PDGFR-β such as Ig-like domain1-3. In some aspect, the other protein binding peptide comprises anextracellular portion of a PDGFR comprising an amino acid sequence ofSEQ ID NO: 11 or an amino acid sequence with at least 85% sequenceidentity to SEQ ID NO: 11.

In accordance with other embodiments, the invention also provides afusion protein comprising an integrin binding peptide that includes anamino acid sequence of SEQ ID NO:1 with at least one mutation on oradjacent to the RGD motif, an amino acid sequence having at least 85%sequence identity to SEQ ID NO: 1 or an amino acid sequence of SEQ IDNO: 2, a human or humanized constant sub-region comprising animmunoglobulin CH2 domain and a CH3 domain, and other protein bindingpeptide having an Ig-like D2 of a VEGFR1 and an Ig-like D3 of a VEGFR2.In a further embodiment of the fusion protein, the integrin bindingpeptide has at least 85% sequence identity to SEQ ID NO: 2.

The term “percent (%) sequence identity” with respect to a referencepolypeptide or nucleic acid sequence is defined as the percentage ofamino acid residues or nucleotides in a candidate sequence that areidentical with the amino acid residues or nucleotides in the referencepolypeptide or nucleic acid sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid or nucleic acid sequence identity can be achieved in variousways that are within the skill in the art, for instance, using publiclyavailable computer software programs, for example, as those described inCurrent Protocols in Molecular Biology (Ausubel et al., eds., 1987), andincluding BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) Software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. For purposesherein, the % amino acid sequence identity of a given amino acidsequence A to, with or against a given amino acid sequence B iscalculated as follows: 100 times the fraction X/Y, where Xis the numberof amino acid residues score as identical matches by the sequencealignment program in that program's alignment of A and B, and where Y isthe total number of amino acid residues in B. It will be appreciatedthat where the length of amino acid sequence A is not equal to thelength of amino acid sequence B, the % amino acid sequence identity of Ato B will not equal the % amino acid sequence identity of B to A.

The present invention provides a dimeric fusion protein comprising twofusion proteins, wherein each fusion protein comprises any fusionprotein disclosed herein. In one embodiment, the dimeric fusion proteincomprises two identical fusion proteins. In another embodiment, thedimeric fusion protein may comprise two different fusion proteins. Thefusion proteins disclosed herein may form multimers of two or moreidentical fusion proteins or form heterologous fusion proteins through amultimerization domain which includes a constant sub-region of a humanor humanized antibody. In some embodiments, the constant sub-region of ahuman or humanized antibody is selected from the group consisting of anIgG Fc region, IgA Fc region, IgM Fc region, IgD Fc region and IgE Fcregion. In the further embodiment, the constant sub-region of a human orhumanized antibody is selected from the group consisting of an IgG1 Fcregion, IgG2 Fc region, IgG3 Fc region and IgG4 Fc region. In someaspect, the sub-region comprises a CH2 region and a CH3 region of IgG1,IgG2, IgG3, or IgG4. Amino acid sequences encoding immunoglobulins thatcomprise Fc regions are well known in the art.

The components of the fusion protein may be connected directly to eachother or be connected via linkers. Generally, the term “linker” meansone or more molecules e.g., nucleic acids, amino acids or non-peptidemoieties which may be inserted between one or more component domains.For example, linkers may be used to provide a desirable site of interestbetween components for ease of manipulation. A linker may also beprovided to enhance expression of the fusion protein from a host cell,to decrease steric hindrance such that the component may assume itsoptimal tertiary structure and/or interact appropriately with its targetmolecule. A linker sequence may include one or more amino acidsnaturally connected to a receptor component, or may be an added sequenceused to enhance expression of the fusion protein, to providespecifically desired sites of interest, to allow component domains toform optimal tertiary structures and/or to enhance the interaction of acomponent with its target molecule.

Preferably, the linker increases flexibility of the fusion proteincomponents without interfering with the structure of each functionalcomponent within the fusion protein. In some embodiments, the linkermoiety is a peptide linker with a length of 2 to 100 amino acids.Exemplary linkers include linear peptides having at least two amino acidresidues such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly (G)n and Gly-Ser(GS) linker. The GS linker described herein includes but is not limitedto (GS)n, (GSGSG)n, (G₂S)n, G₂S₂G, (G₂SG)n, (G₃S)n, (G₄S)n, (GGSGG)nGnand GSG₄SG₄SG, wherein n is 1 or more. One example of the (G)n linkerincludes a G₉ linker. Suitable linear peptides include polyglycine,polyserine, polyproline, polyalanine and oligopeptides consisting ofalanyl and/or serinyl and/or prolinyl and/or glycyl amino acid residues.The linker moieties may be used to link any of the components of thefusion proteins disclosed herein. In some embodiments, a linker is usedbetween an extracellular portion of a receptor protein and a constantsub-region of an immunoglobulin. In other embodiments, a linker is usedbetween disintegrin or its variant and a constant sub-region of animmunoglobulin. In certain embodiments, the fusion protein comprises alinker between an extracellular portion of a receptor protein anddisintegrin or its variant, and a linker between disintegrin or itsvariant and a constant sub-region of an immunoglobulin. As embodied inthe present invention, a fusion protein may comprise at least one linkerbut no more than four linkers.

The fusion protein described herein may or may not comprise a signalpeptide that functions for secreting the fusion protein from a hostcell. A nucleic acid sequence encoding the signal peptide can beoperably linked to a nucleic acid sequence encoding the protein ofinterest. In some embodiments, the fusion protein comprises a signalpeptide. In some embodiment, the fusion protein does not comprise asignal peptide.

Moreover, the fusion proteins described in the present invention maycomprise modified forms of the protein binding peptides. For example,the fusion protein components may have post-translational modifications,including for example, glycosylation, sialylation, acetylation, andphosphorylation to any of the protein binding peptides.

Although the embodiments are generally described with reference to twoprotein binding peptides included in the fusion protein, the inventionalso contemplates a fusion protein which incorporates more than twoprotein binding peptides to provide any additional or synergisticeffects in terms of inhibiting the process of angiogenesis. For example,there may be an additional protein binding peptide that binds to otherangiogenesis targets or acts as angiogenic factor antagonists to belinked to the existing two protein binding peptides.

The present invention provides a nucleic acid encoding a fusion proteinof the invention disclosed herein. The invention further provides amethod of producing a fusion protein disclosed herein. The methodinvolves culturing a host cell transfected with a vector which comprisesa nucleic acid sequence of the invention and recovering the fusionprotein produced by the host cell under suitable conditions.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine or pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases.

Isolated nucleic acid molecules encoding a fusion protein or a componentof a fusion protein can be in the form of RNA, such as mRNA, hnRNA, tRNAor any other form, or in the form of DNA, including, but not limited tocDNA and genomic DNA obtained by cloning or produced synthetically, orany combination thereof. The DNA can be triple-stranded, double-strandedor single-stranded, or any combination thereof. Any portion of at leastone strand of the DNA or RNA can be the coding strand, known as thesense strand, or it can be the non-coding strand, also referred to asthe anti-sense strand. The isolated nucleic acids encoding a fusionprotein or fusion protein component can be prepared by a variety ofmethods known in the art including, but not limited to, isolation from anatural source or preparation by oligonucleotide-mediated mutagenesis,and cassette mutagenesis of an earlier prepared variant or a non-variantversion of the fusion protein or fusion protein component. See MolecularCloning: A Laboratory Manual (Sambrook et al., 4^(th) ed., Cold SpringHarbor, N.Y. 2012) and Current Protocols in Molecular Biology (F. M.Ausubel, et al. eds., 2003).

A “vector” refers to a recombinant plasmid that comprises a nucleic acidto be delivered into a host cell, either in vitro or in vivo.

The present invention contemplates the use of a nucleic acid deliveryvehicle for introduction of one or more nucleic acid sequences encodinga fusion protein or fusion protein component into a cell for expressionof said protein. Examples of nucleic acid delivery vehicles areliposomes, biocompatible polymers, including natural polymers andsynthetic polymers; lipoproteins; polypeptides; polysaccharides;lipopolysaccharides; artificial viral envelopes; metal particles; andbacteria, viruses, such as baculovirus, adenovirus and retrovirus,bacteriophage, cosmid, plasmid, fungal vectors and other recombinantvehicles typically used in the art which have been described in avariety of eukaryotic and prokaryotic hosts. In some embodiments, thenucleic acid delivery vehicle is an expression vector such as a plasmid.The vector may include any element to establish a conventional functionof an expression vector (for example, a promoter, ribosome bindingelement, terminator, enhancer, selection marker), and an origin ofreplication. The promoter can be a constitutive, inducible orrepressible promoter. A number of expression vectors capable ofdelivering nucleic acids to a cell are known in the art and may be usedherein for production of a fusion protein or fusion protein component inthe cell. Expressed fusion proteins or fusion protein components can beharvested from the cells and purified according to conventionaltechniques known in the art and as described herein.

Provided herein are host cells comprising a nucleic acid encoding afusion protein described herein. Nucleic acids encoding fusion proteinsor fusion protein components (e.g., an extracellular portion of anangiogenic factor receptor, a disintegrin or its variant and/or aconstant sub-region of an immunoglobulin) can be provided to a targetcell by any means known in the art. In some embodiments, the nucleicacid encoding a protein of interest is in an expression vector such as aplasmid. In other embodiments, the nucleic acid encoding a protein ofinterest is in a viral vector and the vector has been packaged, then thevirions can be used to infect cells. Transfection and transformationprocedures are known to be appropriately used to introduce a nucleicacid encoding a protein of interest into a target cell. Formulationsutilizing polymers, liposomes, or nanospheres can be used for deliveryof nucleic acids encoding a protein of interest. Cells which aretransformed or transfected with recombinant constructs according to theinvention may be any which are convenient to one skilled in the art.Exemplary cell types which may be used include bacteria, yeast, fungi,insect, plant, and mammalian cells. In some embodiments, transformed ortransfected cells can be provided to a cell or mammalian host. Suitablecells for delivery to a cell or mammalian host include any mammaliancell type from any origin, tumor, or cell line.

Cells used to produce the fusion proteins or fusion protein componentsof the invention are grown in media known to those skilled in the artand suitable for culture of the selected host cells. A given medium isgenerally supplemented as necessary with hormones and/or other growthfactors,

DHFR, salts, buffers, nucleosides, antibiotics, trace elements, andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the cellselected for expression, and will be apparent to one skilled in the art.

Proteins may be purified and identified using commonly known methodssuch as fractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;hydrophobic affinity resins, ligand affinity using a suitable bindingpartner immobilized on a matrix, centrifugation, Enzyme-LinkedImmunosorbent Assay (ELISA), BIACore, Western blot assay, amino acid andnucleic acid sequencing, and biological activity. In some embodiments,the fusion protein is expressed in host cells and purified therefromusing a combination of one or more standard purification techniques,including, but not limited to Protein A affinity chromatography, ProteinG affinity chromatography, buffer exchange, size exclusionchromatography, ultrafiltration, and dialysis. Accordingly, therecovered fusion protein is substantially pure. In a further embodiment,the recovered fusion protein is at least any of 90%, 95%, 96%, 97%, 98%or 99% pure.

The fusion proteins or fusion protein components disclosed herein may becharacterized or assessed for biological activities including, but notlimited to, affinity to a target binding partner, competitive binding,inhibitory activity, inhibition of cell proliferation, inhibition oftumor growth, and inhibition of angiogenesis. In some embodiments, thefusion proteins or fusion protein components disclosed herein can beassessed for biological activity in vitro and in vivo. Many methods forassessing binding affinity are known in the art and can be used toidentify the binding affinities of fusion proteins or fusion proteincomponents to a binding partner. Binding affinities can be expressed asdissociation constant (Kd) values or half maximal effectiveconcentration (EC₅₀) values.

In any of the embodiments herein, a fusion protein has an EC50 of lessthan or equal to 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nMfor inhibition of an activity (e.g., inhibition of angiogenic factoractivity and/or integrin activity). In any of the embodiments herein, afusion protein has a Kd for a binding partner (angiogenic factor and/orintegrin) of less than about 1.0 mM, 500 μM, 100 μM, 50 μM, 10 μM, 5 μM,1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM, 50 pM,10 pM or 5 pM, including any values in between these numbers.

The invention also provides a pharmaceutical composition comprising afusion protein comprising an integrin binding peptide selected from agroup consisting of disintegrin, anti-integrin αvβx antibody,anti-integrin α5β1 antibody, fibronectin targeting integrin αvβx or α5β1and their integrin binding fragments, other protein binding peptidetargeting an angiogenic factor and a Fc domain, wherein x is 1, 3, 5, 6or 8. Compositions of the invention comprise a therapeutically effectiveamount of the fusion protein.

The term “therapeutically effective amount” means an amount of atherapeutically active compound needed to elicit the desired biologicalor clinical effect. According to embodiments of the invention, “atherapeutically effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. Atherapeutically effective amount can be administered in one or moreadministrations. In terms of a disease state, a therapeuticallyeffective amount is an amount sufficient to ameliorate, stabilize, ordelay development of a disease. According to specific embodiments of theinvention, a therapeutically effective amount is an amount of a fusionprotein needed to treat or prevent a disorder characterized by abnormalangiogenesis, such as a disease characterized by neovascularization,vascular permeability, edema, inflammation, retinopathies, fibrosis orcancer.

In some embodiments, the pharmaceutical composition comprising a fusionprotein comprises a fusion protein formulated in a buffer at a proteinconcentration from about 0.5 to about 100 mg/mL, preferably about 40 toabout 80 mg/mL, such as about 40, 50, 60, 70 or 80 mg/mL, mostpreferably about 40±about 5 mg/mL. In other preferred embodiments, thefusion protein is formulated in a buffer at a protein concentration ofmore than about 40 mg/mL.

In particular embodiments, the buffer is a phosphate buffer with a pH ofabout 6.5 to 8, more preferably about 7 to 7.5, even more preferablyabout 7.2. The phosphate buffer comprises about 5 to 20 mM sodiumphosphate, such as 5, 10, 15 or 20 mM sodium phosphate, more preferablyabout 10 mM sodium phosphate; about 20 to 60 mM sodium chloride, morepreferably about 40 mM sodium chloride; about 1 to 10% weight-per-volume(w/v) sucrose, more preferably about 5% w/v sucrose; and about 0.01 to0.05% w/v of a surfactant, more preferably about 0.03% w/v polysorbate20.

In other particular embodiments, the buffer is a histidine buffer with apH of about 5 to 8, more preferably about 6 to 7, most preferably about6.8. The histidine buffer comprises about 10 to 50 mM histidine, such as10, 20, 30, 40 or 50 mM histidine, more preferably about 25 mMhistidine; about 10 to 30 mM sodium chloride, such as 10, 20 or 30 mMsodium chloride, more preferably about 20 mM sodium chloride; about 1 to10% w/v sucrose, such as 1, 2, 4, 6, 8 or 10% w/v sucrose, morepreferably about 6% w/v sucrose; and about 0.01 to 0.05% w/v of asurfactant, more preferably about 0.03% w/v polysorbate 20.

The present invention also relates to a use of the composition accordingto the present invention to treat or prevent an integrin-associateddisease in an individual or a subject. An “individual” or “subject” is amammal. Mammals include, but are not limited to, domesticated animals(e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans andnon-human primates such as monkeys), rabbits and rodents (e.g., mice andrats). In some embodiments, a method of treating or preventing one ormore aspects or symptoms of a disease comprises administering aneffective amount of a composition comprising the fusion protein to anindividual.

The methods described herein can be used for the treatment of a varietyof diseases, including but not limited to, inflammatory disease, oculardisease, autoimmune disease, or cancer. In some embodiments, the diseaseto be treated includes, but is not limited to, rheumatoid arthritis,inflammatory arthritis, osteoarthritis, cancer, fibrosis, retinitispigmentosa, uveitis (such as anterior uveitis or posterior uveitis andocular disease characterized by neovascularization or ischemia (such aschoroidal neovascularization, diabetic retiniopathy, diabetic macularedema, age-related macular degeneration (AMD), retinal vein occlusion,polypoidal choroidal vascularization.

The compositions described herein can be administered to an individualvia any route, including, but not limited to, intravenous,intraperitoneal, ocular, intra-arterial, intrapulmonary, oral,inhalation, intravesicular, intramuscular, intratracheal, subcutaneous,intrathecal, transdermal, transpleural, intraarterial, topical,inhalational, mucosal, subcutaneous, transdermal, gastrointestinal,intraarticular, intracisternal, intraventricular, intracranial,intraurethral, intrahepatic and intratumoral. In some embodiments, thecompositions are administered systemically (for example by intravenousinjection). In some embodiments, the compositions are administeredlocally (for example by intraarterial or intraocular injection).

In some embodiments, the compositions are administered directly to theeye or the eye tissue. In some embodiments, the compositions areadministered topically to the eye, for example, in eye drops. In someembodiments, the compositions are administered by injection to the eye(intraocular injection) or to the tissues associated with the eye. Thecompositions can be administered, for example, by intraocular injection,periocular injection, subretinal injection, intravitreal injection,superchoroidal injection, trans-septal injection, subscleral injection,intrachoroidal injection, intracameral injection, subconjunctivalinjection, sub-Tenon's injection, retrobulbar injection, peribulbarinjection, or posterior juxtascleral delivery. These methods are knownin the art. The compositions may be administered, for example, to thevitreous, aqueous humor, sclera, conjunctiva, the area between thesclera and conjunctiva, the retina choroids tissues, macula, or otherarea in or proximate to the eye of an individual.

The optimal effective amount of the compositions can be determinedempirically and will depend on the type and severity of the disease,route of administration, disease progression and health, mass and bodyarea of the individual. Such determinations are within the skill of onein the art. Compositions comprising a fusion protein can also beadministered six times a week, five times a week, four times a week,three times a week, twice a week, once a week, once every two weeks,once every three weeks, once a month, once every two months, once everythree months, once every six months, once every nine months, or onceevery year.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to describe the methods and/ormaterials in connection with which the publications are cited.

The present invention is further illustrated by the following Examples,which are provided for the purpose of demonstration rather thanlimitation.

Example 1: Production of Rhodostomin Mutant Proteins Method ofDisintegrin Expression

Overlap extension polymerase chain reaction (PCR) techniques were usedto create Rhodostomin (Rho) mutant constructs. The expression kit andthe yeast transfer vector, pPICZaA, were purchased from Invitrogen. Thegene mutant constructs were created by PCR-amplifying the gene sequenceusing a sense primer containing a complementary sequence to the genemutant plus an EcoR1 recognition site and six histidine residues forfacilitating purification, and the antisense primer designed to containa Sac II recognition site and TTA stop codon. The PCR product waspurified and then cloned into the EcoR1 and Sac II sites of yeastrecombination vector, pPICZaA. The recombinant plasmid was transformedinto E coli competent cell DH5a, and colonies were selected by agarplate with low salt Luria Broth (LB, 1% tryptone, 0.5% yeast extract,0.5% NaCl, and 1.5% agar at pH 7.0) and 25 μg/mL of the antibioticZeocin. A single colony was selected to amplify the plasmid forsequencing. After the clones had been confirmed by DNA sequencing, 10 μgof the plasmid was prepared and then linearized by digesting with Sac Ienzyme. The linearized construct was transformed into the Pichia strainX33 using a kit (Pichia EasyComp; Invitrogen), and colonies wereselected by agar plate using YPDS (1% yeast extract, 2% peptone, 2%glucose, 2% agar, and 1 M Sorbitol) and 100 μg/mL of Zeocin. PCRanalysis was used to analyze Pichia integrants to determine whether thegene had integrated into the Pichia genome. From a number of clones withmultiple copies of gene insertion, clones with the highest proteinexpression were chosen.

Method of Disintegrin Purification

Rho mutants were produced as follows: 100 μL of cell stock were grown at30° C. for 48 hours in 200 mL of yeast extract peptone dextrose (YPD)medium (1% yeast extract, 2% peptone, and 2% dextrose) and 100 μg/mLZeocin. Cells were then transferred into 800 mL of YPD medium. Afteranother 48 hours, the cells were collected by centrifugation and grownin 1 L of minimal methanol medium (1.34% yeast nitrogen base (YNB) withammonium sulfate without amino acids, 4×10⁻⁵% biotin, and 1% Methanol).Once every 24 hours, 1% methanol was added to induce protein expressionfor 2 days. The supernatant was collected after centrifugation anddialyzed twice against 5 L of H₂O and once against 5 L of 20 mM Tris-HCland 200 mM NaCl at pH 8. The final solution was loaded into anickel-chelating column and eluted with a gradient of 200 mM imidazole.The recombinant Rho proteins produced in P. pastoris were furtherpurified by reverse-phase C18 HPLC with a gradient of 15% to 18%acetonitrile. A tricine-SDS-PAGE analysis confirmed that thepurification of proteins was greater than 95%.

Example 2: Generation of VEGFR/Rho Mutant Fusion Proteins

The extracellular domain D2 of the Flt-1 receptor (VEGFR1) of SEQ ID NO:8 and the extracellular domain D3 of the Flt-1 receptor (VEGFR2) of SEQID NO: 9, collectively known as VEGF trap, provided the proteincomponent for binding to VEGF ligand. In order to bind to both VEGFligand and integrins, fusion proteins comprising VEGFR1 D2/VEGFR2 D3 ofSEQ ID NO: 10 and Rho mutant of SEQ ID NO: 13 were generated.

A previously generated VEGF trap linked to human immunoglobulin G1 CH2and CH3 domains (IgG1 Fc) was used to generate DNA constructs encodingthe VEGFR1 D2/VEGFR2 D3-Rho mutant hybrid. See Holash, J., et al., PNAS,2002, 99 (17): 11393-11398 for a description of the VEGF trap which isincorporated herein by reference in its entirety. A fusion protein ofSEQ ID NO: 16 was constructed by linking the VEGFR1 D2/VEGFR2 D3 of SEQID NO: 10 and Rho mutant of SEQ ID NO: 13 via at least one peptidelinker of Gly-Gly-Gly-Gly-Ser (G₄S).

Similarly, Fusion Proteins 2 and 3 of SEQ ID NOs: 15 and 17 were alsogenerated by fusing Rho mutants of SEQ ID NO: 12 and SEQ ID NO: 14respectively with the VEGFR1 D2/VEGFR2 D3 of SEQ ID NO: 10. FusionProtein 4 of SEQ ID NO: 18 was generated by fusing a commerciallyavailable VEGF Trap, aflibercept (Eylea®, Regeneron) and Rho mutant ofSEQ ID NO: 13. FIG. 1 provides a schematic showing the structure of afusion protein according to one embodiment of the invention. SS is asignal peptide sequence of SEQ ID NO: 20 which helps secretion ofprotein from the CHO cell. VEGF trap is made up of the extracellularIg-like domains 2 and 3 of human VEGFR1 and VEGFR2. An Fc fragment isthe CH2 and CH3 region of human IgG1. A GS is a linker. Rho mutants havebeen described in U.S. Pat. No. 7,943,728 and PCT Application No. PCT/US15/46322 and their sequences are incorporated herein by reference.

To generate Fusion Protein 1, which has the amino acid sequence of SEQID NO: 16, the DNA sequence encoding Fusion Protein 1 wascodon-optimized for expression in CHO cells. The synthesizedcodon-optimized DNA, with a nucleic acid sequence of SEQ ID NO: 19,together with the signal peptide having a nucleic acid sequence of SEQID NO: 21 was cloned into an expression vector. CHO K1 host cells wereseeded at 2×105 cells/mL in CD CHO (Gibco 12490-003) containing 4 mMGlutamine (J. T Baker 2078-06) and 1% HT Supplement (Gibco 11067-030) 72hours before transfection. The host cells were incubated in an Inforsshaker (36.5° C., 75% humidity, 6% CO₂, 110 RPM) and counted for celldensity before use. A suitable amount of expression plasmid DNA wasadded into the host cells (1 L or 5 L working volume), and polymer-basedtransfection reagent was added. The transfected cultures were incubatedin an Infors shaker (36.5° C., 75% humidity, 6% CO₂, 110 RPM) for 4hours and a proprietary feed solution was added. The transfectedcultures were then incubated in an Infors shaker (32° C., 75% humidity,6% CO₂, 110 RPM). The transfected cultures were harvested on day 10after transfection. The supernatants were purified for generation ofresearch materials. The purification process included clarification,Protein

A affinity chromatography, concentration by Amicon Ultracel, sizeexclusion chromatography, dialysis by Slide-A-Lyzer, and finalconcentration by Amicon Ultracel in the formulation buffer.

Fusion proteins having simultaneous anti-VEGF and anti-integrinactivities were constructed, expressed, purified and characterized. FIG.2 provides a schematic characterizing the purified Fusion Protein 1 bySDS-PAGE. The molecular marker was loaded in Lane M. Non-reduced andreduced forms of engineered VEGF-trap (Positive Control 1; SEQ ID NO:23) consisting of portions of human VEGFR1 and VEGFR2 fused to the Fcportion of human IgG1 were loaded in Lanes 1 and 2, respectively.Non-reduced and reduced forms of commercially available VEGF-trap,aflibercept (Eylea®, Regeneron) (Positive Control 2) were loaded inLanes 3 and 4, respectively. Non-reduced and reduced forms of FusionProtein 1 were loaded in Lanes 5 and 6. Each protein sample was loadedin the volume of 3 μg in each lane. As shown in FIG. 2, the resultsindicate that both the positive control and Fusion Protein 1 exhibitedhigh integrity and purity under reducing and non-reducing conditions.The final concentration of Fusion Protein 1 was approximately 40 mg/mLin the designated formulation buffer (based on the absorption at 280nm).

Production of protein homodimers by cell transfection with theirrespective constructs was confirmed by SDS-PAGE and bioactivityanalysis.

Example 3: Binding Affinity of Fusion Protein to Human VEGF₁₆₅

A direct binding enzyme-linked immunosorbent assay (ELISA) was used tomeasure the binding affinity of fusion proteins of the invention tohuman VEGF₁₆₅, a splice variant of VEGF-A.

VEGF Trap is a soluble VEGF receptor that was engineered for therapeuticuse and is currently approved by FDA to treat AMD. The VEGF Trapcontains the second Ig-like domain 2 (D2) of VEGFR1 fused to the thirdIg-like domain 3 (D3) of VEGFR2 fused to the Fc region of human IgG1(Holash, J., et al, Proc Natl Acad Sci USA. 2002 Aug. 20;99(17):11393-8). VEGF Trap targets VEGF-A, VEGF-B, and PlGF. Thecommercially available VEGF Trap, aflibercept (Eylea®, Regeneron), wasincluded as Positive Control 2.

100 μL of a coating solution (1 μg/mL VEGF₁₆₅ in 1× phosphate bufferedsaline (PBS), pH 7.2) were added to each well of a 96-well ELISA plate,and the plate was incubated overnight at 4° C. The wells were washedtwice with 400 μL of 1×PBS buffer, and excess liquid was carefullyremoved with a paper towel.

400 μL of a blocking solution (5 g non-fat skim milk in 100 mL 1×PBS)were added to each well, and the plate was incubated at room temperaturefor 1 hour. The wells were washed twice with 1×PBS buffer.

Fusion protein and control samples were serially diluted three-fold inblocking solution, with a highest protein concentration of 10 nM. 100 μLof the serially diluted samples were added to each well. The plate wascovered and incubated on a plate shaker (˜100 rpm) for 1 hour at roomtemperature. The wells were washed three times with wash buffer (1×PBS,0.05% Tween-20).

100 μL of 1:2500 diluted horseradish peroxidase-conjugated goatanti-human IgG Fc specific antibodies in blocking solution were added toeach well. The plates were sealed and incubated on a plate shaker for 1hour at room temperature. The plates were washed three times with washbuffer.

100 μL of 3,5,3′,5′-Tetramethylbenzidine (TMB) were added to each well,and the plates were incubated for 3 to 5 minutes to allow for thereaction to take place. To stop the reaction, 100 μL of stop solution(1N HCl) were added to each well.

The optical density (OD) of each well was determined using an ELISAplate reader at an absorbance wavelength of 450 nm. The absorbance wasplotted against the protein concentration of the fusion protein or thecontrol, and the concentration at which the signal was half the maximaleffective concentration (EC₅₀) was determined.

The binding affinity, expressed as the EC₅₀ value, was between 0.10 and0.21 nM for the tested fusion proteins of the invention. The ELISAresults are shown in Table 2

TABLE 2 Test Material EC₅₀ (nM) Positive Control 2 (aflibercept)0.088-0.195 Fusion Protein 1 (SEQ ID NO: 16) 0.106-0.207

Results from Example 3 showed that fusion proteins according toembodiments of the invention, bind VEGF₁₆₅ with high affinity. This isalso illustrated in FIG. 3.

Example 4—Competitive Binding of Fusion Protein to Integrin αvβ3

The competitive binding was used to measure the binding affinity ofFusion Protein 1 of the invention to integrin αvβ3. Wild type Rho (SEQID NO: 1) and Rho variant KG (SEQ ID NO: 13) were synthesized inaccordance with example 1 and included as Positive Control 3 andPositive Control 4 respectively in the competitive binding assay.

1 μg/mL victronectin dissolved in coating solution were added to eachwell of a 96-well ELISA plate, and the plate was incubated overnight at4° C. The wells were washed twice with PBS buffer, and excess liquid wascarefully removed with a paper towel.

Blocking solution (5 g non-fat skim milk in 100 mL 1×PBS) were added toeach well, and the plate was incubated at room temperature for 1 hour.The wells were washed twice with 1×PBS buffer.

Various concentrations of Positive Control 3, Positive Control 4, andFusion Protein 1 were mixed with certain concentration of solubleintegrin αvβ3. One hundred microliters (100 μL) of the serially dilutedsamples were added to each well. The plate was covered and incubated ona plate shaker (˜100 rpm) for 1 hour at room temperature. The wells werewashed three times with wash buffer (1×PBS, 0.05% Tween-20).

Diluted primary anti-integrin αv antibody was added and incubated, thenwashed. Horseradish peroxidase-conjugated goat anti-human IgG Fcspecific antibodies in blocking solution were added to each well. Theplates were sealed and incubated on a plate shaker for 1 hour at roomtemperature. The plates were washed three times with wash buffer.

100 μL of 3,5,3′,5′-Tetramethylbenzidine (TMB) were added to each well,and the plates were incubated for 3 to 5 minutes to allow for thereaction to take place. To stop the reaction, 100 μL of stop solution(1N HCl) were added to each well.

The optical density (OD) of each well was determined using an ELISAplate reader at an absorbance wavelength of 450 nm. The absorbance wasplotted against the protein concentration of the fusion protein or thecontrol, and the concentration at which the signal was half the maximalinhibition concentration (IC₅₀) was determined.

The competitive binding, expressed as the IC₅₀ value, was between 2.2and 16 nM for the tested fusion protein of the invention. The resultsare shown in Table 3 and also illustrated in FIG. 4

TABLE 3 Test Material IC₅₀ (nM) Positive Control 3 (wild type Rho, SEQID NO: 1) 2.6-3.2 Positive Control 4 (Rho variant KG, SEQ ID NO: 13)2.2-2.9 Fusion Protein 1 (SEQ ID NO: 16) 2.2-16.0

Example 5—Inhibition of HUVEC Proliferation by Fusion Protein

A human umbilical vein endothelial cell (HUVEC) proliferation assay wascarried out to test the functionality of the fusion proteins of theinvention. A commercially available VEGF Trap, aflibercept (Eylea®,Regeneron), was included as Positive Control 2.

100 μL of a coating solution (1% gelatin in double distilled water) wereadded to each well of a 96-well ELISA plate, and the plate was incubatedfor 2 hours or overnight at 37° C. The wells were washed twice with1×PBS buffer.

3500 counts of human umbilical vein endothelial cells in endothelialcell growth medium were added to each well, and the plate was incubatedovernight at 37° C.

Fusion protein samples were diluted in assay buffer (Medium-199 1×Earle's Salts, 10% fetal bovine serum, 10 mM HEPES, 1×antibiotic/antimycotic), with a highest protein concentration of 300 nM.The fusion protein samples were mixed with VEGF 165 (8 ng/mL), and themixtures were incubated overnight at room temperature. The wells werethen washed with 200 μL of 1×PBS.

100 μL of the VEGF₁₆₅/sample mixture were added to each well, and theplates were incubated for 72 hours at 37° C. with 5% CO₂. Followingincubation, 10 μL MTS detection reagent(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)+phenazinemethosulfate in distilled PBS) were added to each well, and the plateswere incubated at 37° C. for 2.5 hours.

The OD of each well was determined using an ELISA plate reader at anabsorbance wavelength of 490 nm. The absorbance was plotted against theprotein concentration of the fusion protein or the control, and theconcentration at which the cell proliferation was inhibited by 50%(IC₅₀) was determined.

The inhibition of cell proliferation (IC₅₀) was determined to be between0.039 and 0.103 nM for the tested fusion protein of the invention. Theresults of the proliferation assay are shown in Table 4 and they arealso illustrated in FIG. 5. Both Positive Control 2 and Fusion Protein 1exhibited similar activity on the inhibition of VEGF dependent HUVECproliferation.

TABLE 4 Test Material IC₅₀ (nM) Positive Control 2 (aflibercept)0.026-0.136 Fusion Protein 1 (SEQ ID NO: 16) 0.039-0.103

Example 6—Inhibition of αvβ3 and α5β1 Cell Adhesion by Fusion Protein

The adhesion of αvβ3-overexpressing CHO cells to fibrinogen andα5β1-expressing K562 cells to fibronectin were evaluated in the presenceof fusion proteins.

96-well Immulon-2 microtiter plates (Costar, Corning, N.Y.) were coatedwith 100 μL of phosphate-buffered saline (PBS: 10 mM phosphate buffer,0.15 M NaCl, pH 7.4) containing substrates at a concentration of 50-500nM, and incubated overnight at 4° C. The substrates and their coatingconcentrations were fibrinogen (Fg) at 200 μg/mL and fibronectin (Fn) at25 μg/mL. Non-specific protein binding sites were blocked by incubatingeach well with 200 μL of heat-denatured 1% bovine serum albumin (BSA)(Calbiochem) at room temperature for 1.5 hours. The heat-denatured BSAwas discarded and each well was washed twice with 200 μL of PBS.

CHO cells that expressed the integrin αvβ3 (CHO-αvβ3) from the samesource of Patent Application No. PCT/US2015/46322 were maintained inDMEM. Human erythroleukemia K562 was maintained as written in PatentApplication No. PCT/US2015/46322 from ATCC and cultured in Roswell ParkMemorial Institute (RPMI)-1640 medium containing 5% FCS. Harvested K562was washed in PBS buffer containing 1 mM EDTA and resuspended inTyrode's buffer (150 mM NaCl, 5 mM KCl, and 10 mM HEPES, pH 7.35)containing 1 mM MgSO₄, 2 mM CaCl₂, and 500 μM MnCl₂. Cells (CHO andK562) were diluted to 3×10⁵ cells/mL, and 100 μL of the cells were usedfor the assay. Rho and it mutants were added to the cultured cells andincubated at 37° C., 5% CO₂ for 15 minutes. Rho and its variants wereused as inhibitors at the concentrations of 0.001-500 μM. The treatedcells were then added into the coated plate and incubated at 37° C., 5%CO₂ for 1 hour. The incubation solution was then discarded andnon-adhered cells were removed by washing twice with 200 μL PBS. Boundcells were quantified by crystal violet staining. Briefly, the well wasfixed with 100 μL of 10% formalin for 10 minutes and dried. Fiftymicroliters (50 μL) of 0.05% crystal violet were then added into thewell at room temperature for 20 minutes. Each well was washed with 200μL of distilled water four times and dried. Colorization was carried outby adding 150 μL of colorizing solution (50% alcohol and 0.1% aceticacid). The resulting absorbance was read at 600 nm and the readings werecorrelated with the number of adhering cells.

Inhibition calculation of cell adhesion was conducted according to thefollowing equation.

${\% \mspace{14mu} {Inhibition}} = {100 - {\frac{{{OD}_{600}({TA})} - {{OD}_{600}({NC})}}{{{OD}_{600}({PC})} - {{OD}_{600}({NC})}} \times 100}}$

In the above equation, TA refers to a “test article”; NC refers to“negative control”, and PC refers to “positive control”.

The IC₅₀ was defined as the concentration (nM) of a disintegrin variantrequired for 50% inhibition of the cell adhesion mediated by aparticular integrin. Therefore, lower IC₅₀ indicates greater specificityor potency of the disintegrin variant in inhibiting the cell adhesionactivity of the respective integrin, thus higher binding activity (orselectivity) of the disintegrin variant to the respective integrin. TheIC₅₀ results are summarized in Table 5. FIG. 6 provides a schematicshowing the inhibition of CHO-αvβ3 cell adhesion by Fusion Protein 1.

TABLE 5 Test Material αvβ3, IC₅₀ (nM) α5β1, IC₅₀ (nM) Positive Control 3(SEQ ID NO: 1) 24.7 252.5 Positive Control 4 (SEQ ID NO: 13) 19.6 17.5Fusion Protein 1 (SEQ ID NO: 16) 4.76 3.4

Example 7: Inhibition of HUVEC Tube Formation by Fusion Protein

HUVECs (1.5×10⁴ cells/well) were placed in a 96-well matrigel-coatedplate. Six doses of Fusion Protein 1 (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM,and 30 μM) and vehicle were added to each well in growth media under anatmosphere of 5% CO₂ at 37° C. After an 18-hour incubation period,morphology of the endothelial cell tubes, which resemble acapillary-like network, were evaluated by photomicroscopy. Disruption(anti-angiogenesis) of total tube length was measured from eachphotograph and determined relative to the vehicle control group. Minimuminhibitory concentration (MIC≥30%) of tube formation was then determinedand used to assess the degree of anti-angiogenesis. Suramin was used asan anti-angiogenic positive control (Positive Control 5) for allstudies.

The inhibition of tube formation (IC₅₀) was determined to be less than0.1 μM for the tested Fusion Protein 1 which showed a higher potencythan Positive Control 4. The results of the tube formation assay areshown in Table 6. FIGS. 7A-G provide a schematic showing the inhibitoryeffect of Fusion Protein 1 on tube formation when Fusion Protein 1 wasadded at a dose of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM,respectively.

TABLE 6 Test Material IC₅₀ (μM) Positive Control 5 (Suramin ®) 15.8Positive Control 4 (SEQ ID NO: 13) 5.9 Fusion Protein 1 (SEQ ID NO: 16)<0.1

Example 8—Inhibition of VEGF-Induced Leakage in Dutch Belted Rabbits byFusion Proteins

Fusion proteins of the invention were tested in an in vivo model ofvascular permeability to determine their efficacy in preventing vascularleakage. In this model, VEGF was intravitreally injected to the vitreousof rabbit eyes to induce uncontrolled retinal leakage. Aflibercept(Eylea®, Regeneron), a recombinant fusion protein consisting of portionsof human VEGFR1 and VEGFR2 fused to the Fc portion of human IgG1, andbevacizumab (Avastin®, Roche), a recombinant humanized monoclonalantibody that blocks angiogenesis by inhibiting VEGF-A were included asPositive Controls 2 and 6, respectively.

In one embodiment, a pharmaceutical composition contained 20,000 μg/mLof Fusion Protein 1 in a formulation buffer comprising 25 mM histidinebuffer, 20 mM NaCl, 6% (w/v) sucrose, 0.03% (w/v) polysorbate, pH 6.0.

Dutch Belted rabbits were anesthetized using isoflurane (3-5%), andtheir eyes were treated with ophthalmic Betadine solution. The rabbits'eyes were then washed with sterile saline, and lidocaine hydrochloride(2% injectable) or proparacaine (0.5%) was applied to the ocularsurface.

On Day 1, Dutch Belted rabbits were intravitreally injected with fusionproteins of the invention, vehicle (negative) control, or reference(positive) controls at predetermined doses using a BD 300 μL insulinsyringe (31 ga× 5/16 inch). The needle was inserted through thedorsotemporal quadrant of the eye, approximately 3-4 mm posterior to thelimbus and 3-4 mm lateral to the dorsal rectus muscles, and 50 μL ofsolution was delivered. Vascular leakage was induced by injectingexogenous VEGF₁₆₅ into the same eyes on Day 3.

Fluorescein angiography (FA) was conducted on all dosage groups 3 daysafter VEGF injection to assess leakiness and tortuosity using a scalefrom 0 (normal) to 4 (severe).

Signs of ocular irritation were scored using the Draize scoring systemprior to fusion protein dosing, VEGF induction, and assessments.According to the Draize analysis, all of the rabbit eyes were normalprior to the initiation of dosing. Transient signs of minimal ocularinflammation were observed in all treatment groups after intravitrealdose administration, and were attributed to the intravitreal procedure.There were no drug-related findings evident during the course of thestudy.

FAs associated with the vehicle control group had the highest mean score(2.58) associated with retinal vasculature leakiness and tortuosity. Thetwo reference positive control groups 2 and 6 had mean scores of 0 and0.25, indicating a significant reduction in retinal vasculatureleakiness and tortuosity. The tested fusion proteins of the inventionhad a mean score of 0.167, showing effectiveness in reducingVEGF-induced retinal leakiness and tortuosity comparable to the positivecontrols. The results of the in vivo assay are shown in Table 7. FIGS.8A-D provide the representative FAs showing the inhibition ofVEGF-induced leakage in Dutch Belted Rabbits by the Fusion Protein 1 andpositive controls.

TABLE 7 Dose No. of Day 6 Mean Test Material (μg) Scores Leakage ScoreVehicle 0 12 2.583 Positive Control 6 (bevacizumab) 1250 12 0.250Positive Control 2 (aflibercept) 625 12 0 Fusion Protein 1 (SEQ ID NO:16) 1000 12 0.417

Example 9—Dose-Response Inhibition of VEGF-Induced Leakage in DutchBelted Rabbits by Fusion Protein

Fusion proteins of the invention were tested in an in vivo model ofretinal vascular permeability at varying doses to determine theirdose-response effectiveness in preventing vascular leakage. In thismodel, human VEGF 165 was intravitreally injected to the vitreous ofrabbit eyes to induce retinal leakage.

On Day 1, Dutch Belted rabbits were intravitreally injected with FusionProtein 1 according to an embodiment of the invention at various doses,vehicle (negative) control, or reference (positive) controls. Vascularleakage was induced by injecting exogenous VEGF₁₆₅ into the same eyes onDay 3.

FAs were conducted on all dosage groups 3 days after the VEGF-induction(Day 6) to assess leakiness and tortuosity using a scale from 0 (normal)to 4 (severe).

Signs of ocular irritation were scored using the Draize scoring systemprior to fusion protein dosing, VEGF induction, and assessments.According to the Draize analysis, all of the rabbit eyes were normalprior to the initiation of dosing. Transient signs of minimal ocularinflammation were observed in all treatment groups after intravitrealdose administration, and were attributed to the intravitreal procedure.There were no drug-related findings evident during the course of thestudy.

For the first exogenous VEGF injection, FAs associated with vehiclecontrol group had the highest mean score (3.4) associated with retinalvasculature leakiness and tortuosity. The two reference positive controlgroups had mean scores of 0, indicating a significant reduction inretinal vasculature leakiness and tortuosity. The tested fusion proteinof the invention (Fusion Protein 1) had scores of 0.08, 0.42, and 0.17at doses of 100, 500 and 1000 respectively, showing effectiveness inreducing VEGF-induced retinal leakiness and tortuosity comparable to thepositive controls.

The results of the dose-response in vitro assay are shown in Table 8.

TABLE 8 Dose No. of Day 6 Mean Test Material (μg) Scores Leakage ScoreVehicle 0 10 3.400 Positive Control 6 (bevacizumab) 1250 12 0.17Positive Control 2 (aflibercept) 625 12 0 Fusion Protein 1 (SEQ ID NO:16) 1000 12 0.33 Fusion Protein 1 (SEQ ID NO: 16) 500 10 0.40 FusionProtein 1 (SEQ ID NO: 16) 100 10 0.60

Example 10—Reduction of Lesion Size in Laser-induced ChoroidalNeovascularization (CNV) in Rats by Fusion Proteins

The eyes of Brown Norway rats were dilated with a 1% Cyclogyl solutionand protected from light. Following the dilation, the rats wereanesthetized using a ketamine and xylazine mixture. Three lesion burnswere introduced to the retina of each eye using a laser at 532 nm on Day1.

On Day 3, the animals were anesthetized with a ketamine and xylazinemixture, their eyes were dilated, and 5 μL of Fusion Protein 1, vehicle(negative control), or Positive Control 2 (reference) at predetermineddoses were intravitreally injected into both eyes of an animal using aHamilton syringe with 33 gauge needle.

To confirm successful lesion formation, fundus images were taken using aMicron III small animal funduscope (Phoenix Research) prior to lesionintroduction, after the lesion burns, and on Day 22. The animalsreceived an IP injection of 10% fluorescein sodium at 1 μL/g of bodyweight on Day 22 to assess neovascularization of the lesion burns. Fromfluorescein angiograms, lesion size were determined and compared acrossdosage groups as shown in FIGS. 9A-C, wherein FIG. 9A shows a schematicof vehicle, FIG. 9B shows a schematic of Positive Control 2, and FIG. 9Cshows a schematic of Fusion Protein 1. The arrows (▾) in FIGS. 9A-Cindicated laser lesion spots.

Example 11—Reduction of Lesion Size in Laser-Induced CNV in Monkeys byFusion Proteins

The fusion proteins are tested in a laser-induced CNV model establishedin monkeys. Six to nine burns are introduced around the macula of eacheye using 532 nm diode laser photocoagulation, and 0.5-4 mg of fusionproteins of the invention are intravitreally injected on the same day.

The animals are sedated with intravenous 2.5% soluble pentobarbitone (1mL/kg) 20 days later. The eyelids are fixed to keep the eyes open, andcolor photographs are taken using a fundus camera.

Fluorescein dye (20% fluorescein sodium; 0.05 mL/kg) is injected into avein of a lower extremity. Photographs are taken at several time pointsafter injection of the dye, including the arterial phase, earlyarteriovenous phase, and several late arteriovenous phases, to monitorleakage of fluorescein associated with CNV lesions.

Example 12—Inhibition of Orthotopic Human Glioblastoma Tumor Growth inXenograft Mice by Fusion Proteins

The human glioblastoma cell line U87-MG (2.5×10⁵ cells/2 μL, Luciferasecells) is implanted into BALB/c nude mice to establish an orthotopicxenograft model.

In order to assess the inhibitory effects of the fusion proteins of theinvention on the tumor growth, tumor cells are implanted into nude mice,and various concentrations of fusion proteins according to embodimentsof the invention, ranging from 3 to 30 mg/kg, are administered to themice intravenously twice weekly. The tumor growth, and survival rate ofanimals are measured weekly for up to 8 weeks.

Example 13—Inhibition of Lung Fibrosis in Bleomycin-Induced FibrosisMice by the Fusion Proteins

Male C57BL/6 mice weighing 20±2 g are used to induce lung fibrosis andthe effect of the fusion proteins on inhibiting fibrosis. Animals areanesthetized with isoflurane and then a single dose of bleomycin at 1.5IU/kg (dissolved in 25 μl of saline) is administered intratracheally onDay 1. This dose of bleomycin is known to reproducibly generatepulmonary fibrosis and may induce a mortality of 10-20%.

Nintedanib is administered each day by oral gavage at 10-50 mg/kg perday as a positive control. The administration volume is 10 mL/kg bodyweight. As a negative control, the fusion protein vehicle isadministered to the animals by IV injection. A no treatment control isused to monitor the lung pathology changes after bleomycinadministration. Various doses of Fusion Protein 1 and vehicle areadministered intravenously once a day, starting from Day 1 to Day 21.Clinical observations including body weight are monitored daily. Animalsare sacrificed on Day 22. Bronchoalveolar lavage fluid is collected toinvestigate cell count and TGF-β1.

In order to assess the inhibitory effects of the fusion proteins of theinvention on fibrosis formation, lung tissues are removed andhomogenized in buffer to measure the change of hydroxyproline comparedto a standard curve. Other histopathology experiments are performed togather expression of fibrosis and inflammatory specific biomarkers.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope of the invention.

1.-21. (canceled)
 22. A fusion protein comprising: an integrin bindingpeptide comprising disintegrin binding to integrin αvβx or α5β1 or itsintegrin binding fragments; other protein binding peptide binding to anangiogenic factor, wherein the angiogenic factor comprises Angiopoietin(ANG), Ephrin (Eph), Fibroblast Growth Factor (FGF), Neuropilin (NRP),Plasminogen Activators, urokinase-type plasminogen activator receptor(uPAR), Platelet-Derived Growth Factor (PDGF), Tumor Growth Factor beta(TGF-β), Vascular Endothelial Growth Factor (VEGF), Vascular Endothelialcadherin (VE-cadherin), Insulin-like Growth Factor (IGF),Connective-Tissue Growth Factor (CTGF), Tumor Necrosis Factor alpha(TNF-α), Interleukin 1 (IL-1), Interleukin 6 (IL-6), GranulocyteMacrophage Colony-Stimulating Factor (GM-CSF), and receptors thereof;and a Fc domain; wherein x is 1, 3, 5, 6 or
 8. 23. The fusion proteinaccording to claim 22, comprising the integrin binding peptide, the Fcdomain and the other protein binding peptide from C-terminus toN-terminus or from N-terminus to C-terminus.
 24. The fusion proteinaccording to claim 22, further comprising a linker sequence between theintegrin binding peptide and the other protein binding peptide or asignal peptide sequence upstream of the other protein binding peptide.25. The fusion protein according to claim 22, further comprising a GS orG₉ linker between the Fc domain and the integrin binding peptide orbetween the Fc domain and the other protein binding peptide.
 26. Thefusion protein according to claim 22, wherein the integrin bindingpeptide has at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:7.
 27. A nucleic acid encoding the fusion protein according to claim 22.28. A dimer of the fusion protein according to claim
 22. 29. A vectorcomprising the nucleic acid according to claim
 27. 30. A method ofproducing a fusion protein, comprising culturing a host cell comprisingthe nucleic acid according to claim 27 under a condition that producesthe fusion protein, and recovering the fusion protein produced by thehost cell.
 31. A composition comprising the fusion protein according toclaim 22 and a pharmaceutically acceptable adjuvant, carrier orexcipient.
 32. A fusion protein comprising: an integrin binding peptidehaving an amino acid sequence selected from a group consisting of SEQ IDNO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, and SEQ ID NO: 7, or an amino acid sequence having at least 85%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; other proteinbinding peptide comprising an extracellular domain of a VEGF receptor;and a Fc domain; wherein the integrin binding peptide has at least onemutation on or within 15-20 amino acids from a RGD motif.
 33. The fusionprotein according to claim 32, comprising the integrin binding peptide,the Fc domain and the other protein binding peptide from C-terminus toN-terminus or from N-terminus to C-terminus.
 34. The fusion proteinaccording to claim 32, further comprising a linker sequence between theintegrin binding peptide and the other protein binding peptide or asignal peptide sequence upstream of the other protein binding peptide.35. The fusion protein according to claim 32, further comprising a GS orG₉ linker between the Fc domain and the integrin binding peptide orbetween the Fc domain and the other protein binding peptide.
 36. Thefusion protein according to claim 32, wherein the extracellular domainof the VEGF receptor comprises i) an Ig-like domain D2 of a VEGFR1 andan Ig-like domain D3 of a VEGFR2; ii) the amino acid sequence of SEQ IDNO: 10; iii) an amino acid sequence having at least 90% identity to SEQID NO: 10; or iv) Ig-like domains D1-D7 of the VEGF receptor.
 37. Anucleic acid encoding the fusion protein according to claim
 32. 38. Adimer of the fusion protein according to claim
 32. 39. A vectorcomprising the nucleic acid according to claim
 37. 40. A method ofproducing a fusion protein, comprising culturing a host cell comprisingthe nucleic acid according to claim 37 under a condition that producesthe fusion protein, and recovering the fusion protein produced by thehost cell.
 41. A composition comprising the fusion protein according toclaim 32 and a pharmaceutically acceptable adjuvant, carrier orexcipient.